Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations

Two central features of polymorphonuclear leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic "persistence time" between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an "orientation bias" characterizing the fraction of cells moving up the gradient. A coherent picture of cell movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper, we offer the possibility that "noise" in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we develop a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant/receptor binding. This model can simulate cell paths similar to those observed experimentally, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Furthermore, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. Thus, the concept of signal "noise" can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients.     

Tubulin isotypes and their interaction with microtubule associated proteins

Summary To assay the functional significance of the multiple but closely related - and -tubulin polypeptides (termed isotypes) that are expressed in mammalian cells, we have generated a number of sera that uniquely discriminate among these isotypes. These sera have been used to demonstrate that there is no subcellular sorting of either - or -tubulin isotypes among microtubules of diverse function, either in cells growing in culture or in tissues consisting of cell types that contain specialized kinds of microtubule. In spite of this failure to segregate between functionally distinct kinds of microtubule, the fact that isotype-specific amino acid sequences have been strictly conserved over extensive periods of evolutionary time argues persuasively for a functional role for the different tubulin gene products. One possibility is that they are required for specific interactions with microtubule associated proteins (MAPs), and that tubulin isotypes have coevolved with different cell type-specific MAPs with which they must interact. We have tested this hypothesis by examining the distribution of -tubulin isotypes in mammalian cerebellum in relationship to the known patterns of expression of a number of MAPs, and find that these patterns correlate in the case of M 2 and MAP 3, and M 6 and MAP 1 a. These data, plus emerging data based on a structural analysis of tau, MAP 1 b and MAP 2 obtained via sequence determination of cloned cDNAs, are discussed in terms of the possible functional significance of tubulin isotype/MAP interactionsin vivo.     

Chemotactic peptide binding by rabbit polymorphonuclear leukocytes. Presence of two compartments having similar affinities but different kinetics

N-Formylnorleucylleucylphenylalanine (f-Nle-LeuPhe) bound to rabbit peritoneal polymorphonuclear leukocytes at 4 degrees C exists in at least two compartments that can be differentiated by their off rates. The off rate of one compartment is similar to that of the receptor characterized previously, about 0.4 min-1 (Aswanikumar, S., Corcoran, B., Schiffmann, E., Day, A. R., Freer, R. J., Showell, H. J., Becker, E. L., and Pert, C. B. (1977) Biochem. Biophys. Res. Commun. 74, 810-817; Sullivan, S. J., and Zigmond, S. H. (1980) J. Cell Biol. 85, 703-711); the off rate of the second compartment is about 0.005 min-1. Lysis of the cells at 4 degrees C with 1% Triton does not affect the peptide release from either compartment. Accumulation of peptide at 4 degrees C into the fast off-rate compartment is rapid, reaching a plateau in about 5 min, while peptide in the slow off-rate compartment continues to increase for up to 4 h. The rate of accumulation in the slow off-rate compartment is approximately proportional to the amount of peptide bound to the fast off-rate compartment. Cells lysed at 4 degrees C before binding are still able to accumulate peptide into both compartments. Three possible models to explain the data are presented.     

Parkinson's disease: studies with an animal model

Parkinson' disease has been associated with degeneration of dopamine-containing neurons of the nigrostriatal bundle. Many neurological features of Parkinsonism can be produced in rats by selective destruction of central dopaminergic neurons using the neurotoxin 6-hydroxydopamine. In this review we discuss two aspects of Parkinson's disease that have been investigated in these animals. First, we consider why near-total degeneration of nigrostriatal bundle neurons is required before neurological symptoms emerge. It appears that the loss of dopaminergic neurons is accompanied by an exponential increase in the ratio of tyrosine hydroxylase activity to dopamine content. Thus, after the brain lesions there may be a compensatory increase in the capacity of residual dopaminergic neurons to synthesize and release transmitter. Second, we consider why stress produces severe neurological deficits in patients who are only mildly impaired otherwise. It appears that a variety of stressors produce an abrupt but transient increase in dopaminergic activity in the striatum of intact animals and that this increase is markedly attenuated by 6-hydroxydopamine treatment. Thus, stress-induced akinesia in animals with dopamine-depleting brain lesions and in Parkinsonian patients may result from the impaired ability of residual neurons to respond approximately to such stimuli.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Food acquisition by competing surfperch on a patchy environmental gradient

Synopsis Black surfperch, Embiotoca jacksoni, and striped surfperch, Embiotoca lateralis, coexisted along steep sloping rocky habitats at Santa Cruz Island, California. The range of depths occupied (to 15 m) was characterized by a strong gradient in abundance of prey and a changing mosaic of substrate types from which surfperch harvested food. Availability of prey and diversity of benthic substrates were greatest in shallowest areas and both declined with increasing depth. Individuals of both surfperch species were residential within a narrow range of depths, with the result that different segments of their populations were consistently exposed to different foraging environments. These two phenomena (residential behavior combined with a gradient in availability of resources) resulted in variation in foraging behaviors and diets among individuals that resided at different depths. The pattern of within-population variation differed between the surfperch species. Black surfperch individuals achieved similar taxonomic diets and expended similar foraging effort at all depths, but deep-water foragers captured much less prey biomass per unit effort. The taxonomic composition of striped surfperch diets differed among depths, and although similar amounts of prey biomass were captured everywhere, individuals in deep areas expended much greater effort to obtain that level of food return. For both species, habitat profitability (food return to foraging effort) declined with depth. The difference in habitat profitability appeared to influence fitness components of both surfperches. Individuals occupying deep habitats were about 5% shorter in standard length than conspecifics of the same chronological age living in shallow areas; the disparity in body size resulted in an estimated difference in clutch size of 10–18%.     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.